Effect of topical nitric oxide donors 0.03% nitroglycerin and 0.1% hydralazine on intraocular pressure in healthy canine eyes

Abstract Objective To investigate the potential intraocular pressure (IOP)‐lowering effects of nitric oxide (NO)‐donating compounds in healthy canine eyes Methods A total of 79 dogs were divided into 3 groups in a masked, controlled and randomised study. Group N (n = 26) was administered 0.03% nitroglycerin in one eye and vehicle‐control in the other, Group H (n = 26) was administered 0.1% hydralazine in one eye and vehicle‐control in the other, while Group C (n = 27) received vehicle‐control in both eyes (control group). Following eye drop administration, IOP was measured in both eyes at selected times (10–250 min), along with monitoring of heart rate and signs of ocular discomfort. Data was analysed with repeated measures mixed model and one‐way ANOVA Results IOP was significantly reduced over the 4‐h period with 0.03% nitroglycerin (p < 0.0001) but not 0.1% hydralazine (p = 0.520) when compared to contralateral vehicle‐controlled eyes. IOP was reduced by up to 12% with 0.03% nitroglycerin from 10 to 70 min post‐treatment; however, differences in IOP at individual time points were not statistically significant for either drug (p ≥ 0.133) as compared to contralateral vehicle‐control eyes. No treatment group significantly affected heart rate (compared to Group C), and both treatment groups appeared well tolerated Conclusions Both compounds were well‐tolerated in healthy dogs. Nitroglycerin mildly reduced IOP in canine eyes, and further investigations are warranted in healthy and diseased states (e.g. glaucoma, ocular hypertension).

inhibitors, and beta-blockers (Gelatt et al., 2014). Additional drug classes that have been investigated include alpha2-adrenergic agonists, direct and indirect acting parasympathomimetic agents, calcium channel blockers, and hyperosmotic agents (Gelatt et al., 2014). Current commonly used anti-glaucoma therapies for this disease target one of two mechanisms of IOP regulation: aqueous humor (AH) production or the uveoscleral pathway of AH outflow (Cavet et al., 2014).
Few treatments exist that preferentially influence the conventional pathway of AH outflow, which accounts for up to 85% of AH removal from the normal canine eye (Cavet et al., 2014;Ellis et al., 2009;Miller, 2008). Drug classes also known to largely target the conventional pathway of AH outflow and increase trabecular meshwork outflow facility include Rho Kinase inhibitors and muscarinic agonists (Gelatt et al., 2014;Komaromy et al., 2019). Relevant to this study, nitric oxide (NO)-donating compounds have also been investigated and proven to influence the conventional outflow pathway (Komaromy et al., 2019).
Latanoprostene bunod is the first FDA-approved drug in the category of modified prostaglandin analogues and is composed of latanoprost acid linked to a NO-donating moiety (Addis & Ellis, 2018;Desai et al., 2022;Komaromy et al., 2019). This medication was approved in the United States in 2017 for treatment of openangle glaucoma and ocular hypertension (Desai et al., 2022). The NO-donating moiety, butanediol mononitrate, increases AH outflow through the conventional pathway by relaxing cells in the trabecular meshwork and Schlemm's canal (Burn et al., 2020;Desai et al., 2022).
Latanoprostene bunod does not exclusively affect the conventional outflow pathway, and influences the uveoscleral pathway of AH outflow as well (Komaromy et al., 2019). The current cost of a 2.5 ml bottle is over $200 USD; however, preparation of a solitary ophthalmic NO-donor solution could be a cost-effective option to be used alone or in conjunction with other medications in dogs with glaucoma or post-operative ocular hypertension.
Previous research by Nathanson has explored the efficacy of the individual topical NO donors 0.03% nitroglycerin and 0.1% hydralazine on decreasing IOP in healthy rabbit eyes (Nathanson, 1992). Nathanson's study showed that both compounded drugs were well tolerated in rabbits and reduced IOP in a significant manner, primarily by increasing drainage through the conventional pathway of AH outflow, and to a lesser extent by reducing AH production (Nathanson, 1992). Both drugs are widely available and inexpensive to produce as compounded ophthalmic formulations, so they represent a viable option for canine glaucoma; however, to the author's knowledge, no studies have been performed examining the effects of 0.03% nitroglycerin and 0.1% hydralazine on normal canine eyes.
The aim of this study was to evaluate the effect of the topical NO donors 0.03% nitroglycerin and 0.1% hydralazine on decreasing IOP in the healthy canine eye. Clinical signs of ocular irritation or discomfort were monitored, as well as heart rate to determine whether potential ocular or cardiovascular sequelae to these topical NO-donor formulations would occur in dogs.

Statistical analysis
Normality of data was assessed with the Shapiro-Wilk test. A mixed model for repeated measures was fitted to the data using the R software version 3.6.0, as previously described

DISCUSSION
A great need for effective, widely available, and cost-effective glaucoma therapeutics exists in veterinary medicine. The aim of this study was to determine the efficacy of topical NO donors 0.03% nitroglycerin and 0.1% hydralazine on reducing IOP in healthy canine eyes as potential glaucoma or ocular hypertension therapeutics. Both compounds were well tolerated and did not significantly affect heart rate or cause appreciable signs of ocular irritation. This study showed that 0.03% nitroglycerin was effective in significantly reducing IOP when assessing the entire 4-h trial period. However, neither NO-donor achieved significant reductions in IOP at individual time points when compared to contralateral vehicle-control eyes, potentially due to large variability in IOP measurements. In rabbits, Nathanson and colleagues showed that both nitroglycerin and hydralazine rapidly reduced IOP in a dosedependent fashion, with a peak effect at 1-2 h (Nathanson, 1992).
Differences between rabbits and the present canine study could be explained by species differences in ocular anatomy and physiology . The low blink rate and the thin cornea of rabbits allow for greater drug penetration across the rabbit eye compared to dogs and humans; in fact, a study by Maurice showed that ocular drug exposure is overestimated by threefold in rabbits due to the low blink rate in this species (Maurice, 1995;. Historically, NO donors have been studied for their physiologic effects on the cardiovascular system, immune system, and central and peripheral nervous systems (Cavet et al., 2014;Cavet & Decory, 2018).
In the globe and other tissues of the body, three different isoforms of NO synthase allow for constitutive and inducible production of NO, suggesting basal levels of this molecule may regulate ocular physiology in normal and diseased states (Ellis, 2011). Other studies have more specifically identified NO synthase and NO in canine globes. Samuelson described multiple isoforms of NO synthase in different regions of the normal and glaucomatous canine eye (Samuelson et al., 2001). Additionally, Källberg et al. (2007) identified elevated levels of NO present in the AH and vitreous of normal and glaucomatous canine eyes.
Further studies in veterinary species have shown NO induced ciliary muscle relaxation in bovine and primate species, and decreased cell volume in the porcine trabecular meshwork resulting in increased AH outflow (Dismuke et al., 2008;Gabelt et al., 2011;Wiederholt et al., 1994). No research has been published showing efficacy of NOdonating compounds in canine patients with goniodysgenesis or those with narrow/closed angle glaucoma, as one can speculate that the drug may not alter genetically abnormal trabecular meshwork.
Given NO's many roles, it is possible to imagine that the ocular hypotensive effects of 0.03% nitroglycerin could have been mitigated by coinciding actions on other structures within the eye. To explain NO's bimodal or even hypertensive effects in the eye, Nathanson hypothesised that NO's effects on ciliary body arterial supply caused vasoconstriction and could have resulted in temporarily increased AH production when administered at higher concentrations (Nathanson, 1992). Other possibilities include relaxation of ciliary body smooth muscle in response to NO at higher concentrations, which would simultaneously decrease outflow facility and negate the hypotensive effects of these drugs (Nathanson, 1992). Further work needs to be performed to identify NO's effect on canine intraocular structures to better optimise the efficacy of these drugs.  (Dismuke et al., 2008;Nathanson, 1992). Additionally, recent studies exploring the use of combination therapies (e.g. latanoprostene bunod) showed that combining a drug of known efficacy with a NO-donating moiety could provide synergistic results (Burn et al. 2020;Komaromy et al., 2019;Krauss et al., 2011).
Several ocular hypotensive agents have been proven more efficacious in glaucomatous eyes when compared to normotensive eyes (Gelatt & Mackay, 2001;Gelatt et al., 1979;Gum et al., 1991;King et al., 1991). For example, preliminary studies determined greater hypotensive effects after systemic and topical administration of multiple carbonic anhydrase inhibitors in glaucomatous Beagles than normotensive Beagles (Gelatt et al., 1979;King et al., 1991) The present study used a large and diverse population of dogs, accounting for variability in tear film dynamics that could affect drug bioavailability following eye drop administration (Addis & Ellis, 2018;. However, the study is limited by the lack of a proper acclimatisation phase, recently suggested to extend over a minimum of five training days to enhance IOP data reliability (Fentiman et al., 2019

CONCLUSION
In conclusion, healthy canine eyes that received 0.03% nitroglycerin had a significantly lower IOP over the 4-h testing period when compared to vehicle-control eyes, although the maximal IOP reduction was somewhat limited (12%

ACKNOWLEDGEMENTS
The authors would like to thank pharmacist Dr. Jake Vogel for his assistance with the study. The authors would also like to thank the students, staff, faculty, and their dogs for their voluntary participation.
Open access funding provided by the Iowa State University Library.

CONFLICT OF INTEREST
The authors declare the research was conducted in the absence of any commercial or financial relationships that could be potential conflicts of interest.

ETHICAL AND ANIMAL WELFARE STATEMENT
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. The

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1002/vms3.945